Abstract High mobility group box 1 (HMGB1) is an alarmin actively secreted by immune cells and passively released by necrotic nonimmune cells. HMGB1 has been implicated in both cardiac contractile dysfunction and the lethality associated with sepsis/endotoxemia. The aim of the current study was to assess whether viable cardiomyocytes could produce HMGB1 and whether HMGB1 can affect myocardial contractility. LPS was used as a model of sepsis/endotoxemia in mice and isolated cardiac myocytes. LPS increased myocardial expression of HMGB1 in vivo (immunohistochemistry) and production and secretion of HMGB1 by viable cardiac myocytes in vitro (Western). LPS increased the phosphorylation status of PI3Kγ in cardiac myocytes, an effect not observed in TLR4−/− myocytes. Genetic (PI3Kγ−/−) or pharmacologic (AS605240) blockade of PI3Kγ ameliorated the LPS-induced 1) cardiomyocyte production and secretion of HMGB1 in vitro and 2) HMGB1 expression in the myocardium in vivo. The LPS-induced depression of myocardial contractility was prevented by the HMGB1 antagonist, A-box. Genetic (PI3Kγ−/−) or pharmacologic (AS605240) blockade of PI3Kγ ameliorated the LPS-induced decrease in myocardial contractility. No evidence of inflammatory infiltrate was noted in any of the in vivo studies. The findings of the current study indicate that 1) LPS can induce HMGB1 secretion by viable cardiac myocytes through a TLR4/PI3Kγ signaling pathway, and 2) HMGB1 plays a role in the LPS-induced myocardial contractile dysfunction. The results of the current study also have broader implications (i.e., that viable parenchymal cells, such as cardiac myocytes, participate in the alarmin response).